This invention relates to the fields of molecular biology and peptide transporters. Specifically, nucleic and amino acid sequences for expressing the human peptide histidine transporter 1, (hPHT1) and fragments thereof as well as hPHT1 immunospecific antibodies are provided. Methods and kits employing the compositions of the invention are also disclosed.
Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Peptide transport is a specific biochemical process in which small peptides are transported across a membrane by energy-dependent saturable carriers. While the existence of a transport process for intact peptides in the brush border membrane of intestinal and renal absorptive epithelial cells has been known for almost three decades, it is only recently that the molecular nature of the proteins responsible for the transport process has been elucidated. Two peptide transporters, PEPT 1 and PEPT 2, have been cloned. The cloned transporters catalyze active transport of intact di- and tripeptides and utilize a transmembrane electrochemical H+ gradient as the driving force. The characteristic of H+ coupling makes PEPT 1 and PEPT 2 unique among the transporters thus far identified in mammalian cells. In addition, the peptide transporters have immediate pharmacologic relevance because a number of peptide-like drugs are recognized as substrates by these transporters. Recently, cultured cell lines of intestinal and renal origin that express PEPT 1 and PEPT 2 have been identified. (Oh et al., 1999, Pharm Biotechnol. 12:59-88; Liang et al., 1995, J Biol Chem. 270:6456-6463; Fei et al., 1994, Nature 368:563-566; Dantzig et el., 1994, Science 264:430-433).
PepT1 belongs to the proton oligopeptide transporter (POT) superfamily, in which all of the known peptide transporters, with the exception of HPT1 (a cadherin family member), are grouped (Meredith and Boyd, 2000, Cell Mol Life Sci. 57:754-778; Graul and Sadee, 1997, Pharm. Res. 14(4):388-400). Of these transporters, PepT1 has been extensively characterized and appears to be the predominant peptide transporter in the gastrointestinal tract (GIT) (Pauletti et al., 1996, J Cont Rel. 41:3-17; Oh et al., 1999, Pharm Biotechnol. 12:59-88; Ganapathy et al., 1998, Biochem Biophys Res Commun. 246:470-475; Tamai et al., 1997, J Pharm Pharmacol. 49:796-801). More recently, HPT-1 has been identified in Caco-2 cells (Dantzig et el., 1994, Science 264:430-433; Hu et al., 1994, Pharm Res. 11:1405-1413; Hu et al. 1995, Pharm Res. 12:1120-1125; Snyder et al., 1997, Antimicrob Agents Chemother; Yang, 1998, thesis, Purdue University, West Lafayette, Ind.).
Inasmuch as the therapeutic use of peptide-based pharmaceuticals is a burgeoning area in rational drug design, elucidation of the specificity and molecular function of different transporter molecules is highly desirable for the treatment of a variety of pathological disorders.
The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or variants thereof, that participate in peptide transport within the GIT.
According to one aspect of the invention, an isolated nucleic acid molecule is provided which includes a complete coding region for a human peptide histidine transporter 1 (hPHT1)protein of a predicted size between about 50 and 80 kilodaltons. The hPHT1 protein is preferably about 65 kilodaltons. As predicted by structural analysis, the hPHT1 protein comprises twelve putative transmembrane domains (TM) and includes NH2xe2x80x94 and COOH terminal ends that are localized to the cytoplasm of cells.
In yet another embodiment of the invention, an isolated nucleic acid encoding a hPHT1 protein is provided. In a particularly preferred embodiment, the hPHT1 protein has an amino acid sequence the same as Sequence I.D. No. 2. An exemplary hPHT1 encoding nucleic acid molecule of the invention comprises Sequence I.D. No. 1.
In another embodiment of the invention, an isolated nucleic acid encoding a splice variant of hPHT1 protein is provided. In a particularly preferred embodiment, a splice variant of hPHT protein has an amino acid sequence the same as Sequence I.D. No. 4. An exemplary hPHT1 splice variant encoding nucleic acid molecule of the invention comprises Sequence I.D. No. 3.
In yet another embodiment of the invention, variant hPHT1 encoding nucleic acids have been isolated. These nucleic acid sequences are referred to herein as SEQ ID NOs: 53 and 55. The amino acid sequences encoded by SEQ ID Nos: 53 and 55 are referred to herein as SEQ ID NOs: 54 and 56.
According to another aspect of the invention, antibodies immunologically specific for the proteins described hereinabove are provided.
In addition, this invention presents methods for screening potentially beneficial therapeutic agents which modulate hPHT1 meditated transport. Agents so identified are also within the scope of the present invention.